Panic disorder (PD) is a severe anxiety disorder characterized by recurrent panic attacks affecting about 2- 5% of the population and resulting in severe disability in about a third of those subjects. During the previous funding period, using our well established animal model we identified that orexin (hypocretin) neurons within the perifornical-dorsomedial hypothalamic region (PeF/DMH) are one of the key regulators of a coordinated panic response and that patients with panic but not depression symptoms did indeed have high levels of orexin in their cerebrospinal fluid. Little is known about how this disrupted panic regulation leads to many of its debilitating consequences such as chronic anticipatory anxiety and agoraphobia. Therefore, the goal of this competitive renewal (MH 52619-14 to -19) is to elucidate the neural mechanisms involved in how a panic- vulnerable state also leads to increased chronic anxiety and enhanced vulnerability to conditioned fears, avoidance and phobias. The project will have significant impact as we hope that the results of these studies will identify novel therapeutic targets for not only panic attacks, but also their many disabling consequences such as agoraphobia. Specifically, we predict that chronic disinhibition of orexinergic neurons in the PeF/DMH results in disruption of neurotransmission of not only orexin, but also its co-transmitters glutamate and dynorphin, resulting in disrupted network functions in 1) the bed nucleus of the stria terminalis (BNST); 2) the central nucleus of the amygdala (CeA); and 3) the infralimbic prefrontal cortex (IL) to delay the extinction of conditioned fear. We will elucidate these mechanisms with behavioral, molecular and electrophysiological endpoints, utilizing pharmacological, gene silencing, optogenetic experiments in both whole animal and slice preparations. Specific Aim 1 will focus on chronic disinhibition of ORX neurons in the DMH/PeF inducing molecular and network changes in the BNST to decrease GABAergic and enhance glutamatergic neurotransmission to induce a chronic anxiety-like phenotype. Specific Aim 2 will study the mechanisms within the CeA that result in enhanced acquisition and expression of conditioned fears seen in panic-prone rats, testing if chronic disinhibition of ORX neurons in the DMH/PeF enhances excitation and reduces inhibition, thus facilitating neuronal plasticity in the CeA. In Specific Aim 3, we propose to stud the mechanisms within the IL which could result in delayed extinction of conditioned fear responses in panic-prone rats. This project is innovative at several level providing a mechanistic model to understand the pathophysiology of PD and its disabling consequences combining the study of whole animal behavior, physiology, and systems level pharmacological and optogenetic manipulations with functional network electrophysiology as well as basic cellular and molecular changes. Finally, at the translational level, the work has already had significant impact in the development of novel therapies for acute panic attacks, but hopefully will provide novel insights into the persistent and hard to extinguish nature of agoraphobia and anticipatory anxiety in patients with panic disorder.